Insect pests cost the general public billions of dollars annually in losses. These losses include the expense of controlling insect pests as well as crop loss and property damage caused by the pests. Economically significant insect pests in United States include Solenopsis spp. (fire ants) and moth species, such as Helicoverpa zea (commonly referred to as the cotton bollworm, corn earworm, or tomato fruitworm, depending on the plant the larvae are feeding on). For Solenopsis invicta specifically, the well-documented fire ant currently infests over 320 million acres in the United States and over $6 billion per year is spent for control, agricultural losses, medical costs, and damage repair (as reported in Lard, C. F., J. Schmidt, B. Morris, L. Estes, C. Ryan, and D. Bergquist. 2006. “An economic impact of imported fire ants in the United States of America.” Texas A&M University, College Station, Tex. Available online at http://fireantecon.tamu.edu). Control of Solenopsis invicta is generally achieved through traditional chemical pesticides and/or delayed acting pesticides delivered as baits.
The insect pest Helicoverpa zea also has the capacity to inflict devastating yield losses to over 100 crop plants, the most important of which are corn, cotton, tomato, bean, broccoli, cabbage, eggplant, lettuce, okra, pea, pepper, soybean and watermelon. Due to its high multiplication rate, H. zea can rapidly build up large populations, so the feeding caterpillars can cause devastating crop losses. Helicoverpa zea is typically controlled through the use of pyrethroid and Bacillus thuringiensis (Bt) insecticides.
Chemical pesticides are the primary tools used to combat these insect pests. However, use of traditional chemical pesticides has disadvantages, including non-target effects on neutral or beneficial insects, as well as other animals. Chemical pesticide usage also can lead to chemical residue run-off into streams and seepage into water supplies resulting in ecosystem/environment damage. In addition, animals higher in the food chain are at risk when they consume pesticide contaminated crops or insects. The handling and application of chemical pesticides also presents exposure danger to the public and professionals, and could lead to accidental dispersal into unintended environmentally sensitive areas. In addition, prolonged chemical pesticide application may result in an insect population becoming resistance to a chemical pesticide. In order to control a traditionally chemical resistant-pest, new more potent chemical pesticides must be utilized, which in turn will lead to another resistance cycle. As such, there is a need in the art to control pest populations without the disadvantages of traditional chemical pesticides.
An approach to decrease dependence on chemical pesticides is by causing a specific gene(s) of the target-pest to malfunction by either over expression or silencing gene expression. The silencing approach utilizes RNA interference pathways to knockdown the gene of interest via double strand RNA. Double strand RNA (dsRNA) induces sequence-specific post-transcriptional gene silencing in many organisms by a process known as RNA interference (RNAi). RNAi is a post-transcriptional, highly conserved process in eukaryotes that leads to specific gene silencing through degradation of the target mRNA. The silencing mechanism is mediated by dsRNA that is homologous in sequence to the gene of interest. The dsRNA is processed into small interfering RNA (siRNA) by an endogenous enzyme called DICER inside the target pest, and the siRNAs are then incorporated into a multi-component RNA-induced silencing complex (RISC), which finds and cleaves the target mRNA. The dsRNA inhibits expression of at least one gene within the target, which exerts a deleterious effect upon the target.
Fire, et al. (U.S. Pat. No. 6,506,559) discloses a process of introducing RNA into a living cell to inhibit gene expression of a target gene in that cell. The RNA has a region with double-stranded structure. Inhibition is sequence-specific in that the nucleotide sequences of the duplex region of the RNA and of a portion of the target gene are identical. Specifically, Fire, et al. (U.S. Pat. No. 6,506,559) discloses a method to inhibit expression of a target gene in a cell, the method comprising introduction of a double stranded ribonucleic acid into the cell in an amount sufficient to inhibit expression of the target gene, wherein the RNA is a double-stranded molecule with a first ribonucleic acid strand consisting essentially of a ribonucleotide sequence which corresponds to a nucleotide sequence of the target gene and a second ribonucleic acid strand consisting essentially of a ribonucleotide sequence which is complementary to the nucleotide sequence of the target gene. Furthermore, the first and the second ribonucleotide strands are separately complementary strands that hybridize to each other to form the said double-stranded construct, and the double-stranded construct inhibits expression of the target gene.
To utilize RNA interference as a method to regulate gene expression for control, a specific essential gene needs to be targeted. Genes associated with signal transduction of neurohormones represent novel potential targets. One neurohormone gene family is the pheromone-biosynthesis-activating neuropeptide receptor (PBAN-R) gene family. Each PBAN-R is expressed in target tissue(s) in developmental and adult stages. The PBAN-R activates a specific physiological function after binding to a PBAN/pyrokinin peptide ligand. To date, over 200 PBAN/pyrokinin family peptide ligands have been identified. These peptide ligands/receptors have been shown to have a variety of functions in insects, such as: 1) stimulate pheromone biosynthesis in female moths (Raina et al., 1989); 2) induce melanization in moth larvae (Matsumoto et al., 1990; Altstein et al., 1996); 3) induce embryonic diapause and seasonal polyphenism in moths (Suwan et al., 1994; Uehara et al., 2011); 4) stimulate visceral muscle contraction (Nachman et al., 1986; Predel and Nachman, 2001); 5) accelerate puparium formation in several flies (Zdarek et al., 1997; Verleyen et al., 2004); 6) terminate pupal diapause in heliothine moths (Sun et al., 2003; Xu and Denlinger, 2003).
There is a need in the art to determine whether dsRNA interference of PBAN receptor expression interferes with essential developmental and/or reproductive functions of targeted insect pests and results in abnormal development and/or lack of reproduction. It has been reported in U.S. patent Ser. No. 13/323,880 (Vander Meer et al.) and Ser. No. 13/324,005 (Vander Meer et al.) that double-stranded ribonucleic acid construct that is complementary to a gene that encodes a PBAN/Pyrokinin gene sequence has a controlling effect on insects, e.g. Solenopsis invicta and Helicoverpa zea. However, there is a further need to determine whether dsRNA silencing of a PBAN receptor gene has an effect on controlling insects.
Furthermore there is a need for novel control methods that would interfere with essential developmental and/or reproductive functions of species that do not have the undesirable characteristics of traditional chemical pesticides. To that end, there is a need to develop dsRNA constructs that are engineered to interfere with essential developmental and/or reproductive functions of specific pest insects that would overcome some of the disadvantages of using traditional pesticides, specifically dsRNA interference of PBAN receptor gene expression.